Described herein is a chip-based apparatus for amplifying nucleic acids, a cartridge housing the apparatus, and methods of using the apparatus for amplification of nucleic acids. More specifically, this invention provides integrated circuit semiconductor chip, manufactured with standard semiconductor manufacturing process, to perform thermal management and optical sensing necessary for on-chip amplification and detection of nucleic acids. The apparatus and methods embodied in this invention make it possible to build a disease diagnosis and prognosis tool that is easy to use, portable and disposable.
The detection and quantification of nucleic acid is becoming more and more important in: (1) medicine, specifically disease diagnosis or prognosis and drug discovery; (2) crop and animal breeding and authentication; (3) forensic identification; 4) environmental monitoring and industrial processing. The Polymerase Chain Reaction, or PCR, is a method for replicating a nucleic acid of interest many times. PCR is widely used in detecting small amounts of nucleic acids in a sample. It is particularly useful for the detection of diseases, including infectious diseases and cancer.
Real time PCR, also known as qPCR, allows the accumulation of PCR-amplified nucleic acids to be monitored in real time instead of at the end point of the reaction. Real time monitoring of the buildup of PCR products allows one to better manage the reaction and quantify the concentration of the target nucleic acid.
Conventional instruments for real time PCR are typically bulky and costly. Examples are BioRad iCycler®, Life Technologies StepOne® real time PCR system, Roche Diagnostics LightCycler® 2.0, Qiagen's Roto-Gene® system. The reasons are twofold. The conventional thermal management method depends on a structure with large thermal capacity to achieve precise control of temperature. Typically this is achieved through the use a sizable metal heating block and a cooling reservoirs which regulate the temperature of the samples contained in plastic tubes.
Moreover, to achieve real time monitoring of PCR product accumulation, optical systems are involved to detect optically labeled target molecules. In a conventional design, optical monitoring is achieved through use of many discrete components, such as photo multipliers (PMT, a type of vacuum tube), discrete photodiodes or CCD sensors. Optical excitation is achieved through the use of lamps, laser diodes or high power LEDs. These components and the associated lenses, filters and mechanical structures typically require substantial space and diminish the portability of the associated apparatus.
PCR devices are typically designed to house multiple “wells”, where samples contained in tubes are placed. This design is necessary to perform PCR on many different samples, some of which are negative or positive controls. To perform optical detection of many samples with optical detection systems made of discrete components typically requires a motion control system to move sample tubes individually into the optical detection pathway. Again, this typical design increases the complexity, size and cost of the PCR device. It also introduces an additional artifact: detection times are different for different samples. This artifact reduces sample-to-sample reproducibility.
Efforts have been made to allow PCR to occur on a “chip”, with very small reaction volumes (e.g., WAFERGEN's SmartChip™ and BECKMAN COULTER's AmpliGrid™ system). However, like many biological lab-on-chip systems, these “chips” are nothing more than a passive substrate made of plastic and/or glass. These “chips” alone cannot perform the necessary functions for PCR. In order for PCR reactions to occur, these “chips” need to be placed into a bulky thermal cycler much like the conventional ones described above. Many of these “chip”-based thermal cyclers in the market are in fact bigger and more expensive than the mainstream tube based thermal cyclers. At the end of the reaction, the results of typical “chip”-based PCR systems are generally detected or monitored with the assistance of a bulky and expensive optical system such as a fluorescent microscope.
Each year, a large population of the world is affected by outbreaks of various types of infectious diseases, such as SRS, influenza/H1N1/H5N1, foot-and-mouth disease, TB, HBV, HCV, HIV, etc. Many of these diseases, at least at the onset, are treatable. However, the lack of health care in many poorer regions of the world coupled with a dense population causes many instances of disease to go undiagnosed, untreated or mistreated. Untreated or mistreated pathogens spread, mutate and evolve into pandemics, affecting the lives of millions and billions of dollars. A need therefore exists for effective diagnostic tools that are easy to use, low cost, portable and disposable, such that the diagnostic procedures can be reliably performed at the point-of-care.